Khd1p, a protein with multiple roles in mRNA localization and telomeric silencing [Elektronische Ressource] / Gonçalo Nuno Nunes da Silva Rebelo de Andrade

Goncalo Rebelo De Andrade

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 16 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultaet fuer Chemie und Pharmazie
der Ludwig-Maximillians-Universitaet Muenchen











Khd1p, a protein with multiple roles in mRNA localization and
Telomeric Silencing










Gonçalo Nuno Nunes da Silva Rebelo de Andrade

aus

Lissabon





2008
Erklaerung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29.
Januar 1998 von Herrn Professor Doktor Ralf Peter Jansen betreut.





Ehrenwoertliche Versicherung

Diese Dissertation wurde selsbtaendig, ohne unerlaubte Hilfe erarbeitet.


Muenchen, am 20. Juni 2008








__________________________________________









Dissertation eingereicht am 24. Juni 2008

1.Gutachter Herr Professor Doktor Ralf-Peter Jansen
2.Gutachter Herr Professor Doktor Klaus Foerstemann

Muendliche Pruefung am 28. Juli 2008 Table of Contents
1. INTRODUCTION ........................................................................................................................... 8
1.1 Mechanisms of mRNA localization .......................................................................................... 8
1.2 Conceptual Mechanism of cytoplasmic mRNA localization by active transport ..................... 9
1.3 From the birth of a transcript to nuclear export of a mature mRNA......................................... 9
1.4 Reaching the cytoplasm .......................................................................................................... 10
1.5 mRNPs travelling along the cytoskeleton ...............................................................................11
1.5.1 Role of the cytoskeleton....................................................................................................... 12
1.5.2 Role of the molecular motor binding adaptor proteins in determining cargo specificity .... 12
1.6 Reaching the destination ......................................................................................................... 13
1.7 mRNA Localization in S. cerevisiae....................................................................................... 14
1.8 ASH1 mRNA localization.. 15
1.9 ASH1 mRNA .......................................................................................................................... 16
1.10 Translation Regulation .......................................................................................................... 17
1.11 The core localization machinery ...........................................................................................18
1.12 Accessory factors .................................................................................................................. 19
■ LOC1...................................................................................................................................... 19
■ PUF6....... 20
■ SCP160... 20
■ KHD1...... 21
1.13 Aim of this work ................................................................................................................... 21
2. MATERIAL................................................................................................................................... 22
2.1 PLASMIDS LIST:................................................................................................................... 22
2.2 YEAST STRAINS:.... 24
2.3 OLIGONUCLEOTIDES LIST ............................................................................................... 30
2.4 General Laboratory Material................................................................................................... 34
2.4.1 Laboratory equipment and disposables ........................................................................... 34
3. METHODS .................................................................................................................................... 37
3.1 Bacteria.................................................................................................................................... 37
3.1.1 Preparation of E. coli competent cells............................................................................. 37
3.1.2 E. coli transformation ...................................................................................................... 37
3.1.3 Preparation of Electro-Com..........................................................................37
3.1.4 Transformation of Electro-Competent cells. ................................................................... 38
3.2 Eukarya.................................................................................................................................... 38
3.2.1 Transformation protocols ................................................................................................ 38
3.2.2 Determining mRNA half-life: ......................................................................................... 39
3.2.3 Determining protein content............................................................................................ 43
3.2.4 Tandem Affinity Purification (TAP) ............................................................................... 44
3.2.5 Determining Telomeric Silencing ................................................................................... 46
3.2.6 FACS ............................................................................................................................... 47
3.2.7 Immunofluorescence ....................................................................................................... 48
3.2.8 UltraViolet CrossLinked Immuno-Precipitation (CLIP).................................................48
3.2.9 Flotation Assay................................................................................................................ 52
3.2.10 Yeast Colony PCR......................................................................................................... 53
3.2.11 Immunoprecipitation .....................................................................................................53
3.2.12 Point mutant generation.................................................................................................54
3.2.13 Polysome profiling analysis...........................................................................................55
4. RESULTS ...................................................................................................................................... 57
4.1 Relationship of ASH1 mRNA localization and nonsense mediated decay (NMD)?.............. 57
4.2 Relationship between RNA localization factors and cell viability?........................................59
4.3 What is Khd1p connection to ASH1 mRNA localization process? 59
4.4 Which co-purifying proteins can be found with this approach? ............................................. 60
4.5 Is Khd1p a part of a RNA dependent protein complex? ......................................................... 61
4.6 Is Khd1p a part of the locasome? Can it co-purify other members of the ASH1 mRNP?......62
4.6.1 Does it also co-purify other members of the locasome? .................................................62
4.6.2 Is there a direct interaction between She3p and Khd1p? 63
4.7 Khd1p, another mRNA binding protein associated to the Endoplasmic Reticulum (ER)? ....65
4.7.1 Does Khd1p-affinity purification also yield ER-bound proteins?................................... 66
4.8 Khd1p proposed to be a translation repressor. Does it show translation inhibitor sensitivity?66
4.9 Is Khd1p associated with polysomes?..................................................................................... 67
4.10 Khd1p, an mRNA binding protein? ...................................................................................... 70
4.11 Ultraviolet CrossLinked ImmunoPrecipitation (CLIP)......................................................... 71
5. DISCUSSION ................................................................................................................................ 72
5.1 Is there a relationship between ASH1 mRNA localization and nonsense mediated decay
(NMD)? ......................................................................................................................................... 72
5.2 Relationship between RNA localization factors and cell viability?........................................74
5.3 What is Khd1p connection to ASH1 mRNA localization process? 75
5.4 Khd1p, another mRNA binding protein associated to the Endoplasmic Reticulum (ER)? ....77
5.5 A new model for ASH1 mRNP architecture...........................................................................78
5.6 Khd1p has been proposed to be a translation repressor. ......................................................... 80
5.6.1 Does it show translation inhibitor sensitivity? ..................................................................... 80
5.6.2 Is Khd1p associated with polysomes?.................................................................................. 80